The human immune system is composed of several tissues that are enriched with white blood cells (WBCs) including bone marrow and lymph nodes. WBCs participate in two layers of immune defense, the innate immune response and the adaptive immune response. The innate immune response is the first line of immune defense and is primarily made up of WBCs from the myeloid lineage including neutrophils, monocytes, eosinophils and basophils. These are early responding cells that stage an initial defense and alert the rest of the immune system of an infection. The second line of immune defense is the adaptive immune system and is primarily made up of WBCs from the lymphoid lineage including B cells and T cells. These cells wait to receive information about the invading pathogen and then mount a targeted response.
Natural Killer (NK) cells are specialized lymphocytes that act in innate immunity. They are critically important in the anti-viral response and patients lacking NK cells have persistent viral infections. NK cells destroy compromised cells by recognizing abnormally low levels of major histocompatibility complex (MHC) I. This capability also makes NK cells potent against tumor cells which similarly lack MHC I markers. NK cells mature in the bone marrow and other lymph tissues then enter circulation in blood. From the blood NK cells seek infected or oncogenic tissues by following a trail of inflammatory cytokines secreted by monocytes and other early responding cells. Exposure of NK cells to cytokines such as interferon (IFN) alpha (α), IFN beta (β), and Interleukin 2, 12, 15, 18 and 21 as well as Tumor Necrosis Factor alpha (TNF a) increases NK cytotoxicity by orders of magnitude (Sivori et al. 2004). Such cytotoxic NK cells respond aggressively by killing infected cells and thereby limiting the spread of infection.
Using lymphocytes for adoptive transfer therapy was first reported over 50 years ago where transplanted T cells conferred immunity to cancer in rodent models (Mitchison 1955). Adoptive T cell transfer involves the isolation of T cells from blood or bone marrow followed by concentration or expansion of the cells in vitro. Once a sufficiently large or concentrated population is obtained the T cells are infused into a patient (Restifo et al. 2006, Kalos et al. 2013). More recently clinicians have sought to use NK cells for adoptive transfer because of their ability to recognize and kill tumor cells without requiring any particular tumor cell marker (Alizadeh et al. 2010). However development of NK cell adoptive transfer procedures has been impeded by the limited supply of viable cells. NK cells represent only a small fraction of the cells in blood and isolation from a typical blood draw does not yield many cells. Furthermore, NK cells must be purified away from contaminating PBMCs such as T and B cells by CD3 and CD19 depletion, respectively (Childs et al. 2013). This is a necessary step for allogenic transplantation where the presence of T and B cells increases risk of graft versus host disease (GVHD) but further reduces the NK cell yield.
In addition, NK cells expand poorly in vitro compared to others kinds of cells due mainly to early senescence (Childs et al. 2013, Denman et al. 2012). Using even the most effective methods, NK cells are susceptible to telomere shortening and senescence after only a few passages (Denmon et al. 2012). The most effective method for increasing NK cell viability and proliferation in vitro is co-culturing with feeder cells. Commonly used feeder cells for NK expansion include irradiated peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed lymphoblastoid cell lines (EBC-LCL), gene-modified K562 cells constitutively expressing IL-15 or 21, and other irradiated tumor cell lines (Berg et al. 2009, Childs et al. 2013, Baek et al. 2013). Co-culturing with feeder cells significantly increases NK cell viability and proliferation with population increases between 1,000 and 50,000 times (Denman et al. 2012, Childs et al. 2013). Although NK cells grown on feeder cells can be used clinically, feeder cells remain undesirable because of the increased risk of contamination and the need for additional testing for bacteria, endotoxin and mycoplasma contamination (Childs et al. 2013).
NK cells may be cultured without feeder cells if provided with sufficient cytokines such as IL-2, 12, 15, 18, 21 or nicotinamide. The resulting NK cells exhibit increased cytotoxicity compared to freshly isolated NK cells but can only be expanded between 100 and 300 times (Carlens et al. 2001, Childs et al. 2013, Klingeman et al. 2004). The limited expansion potential is due to telomere loss and senescence (Denman et a. 2012). In addition, residual IL-2 can have severe side effects on patients (Ni 2013).
A problem related to the short supply and difficulty of expanding NK cells is the fact that they do not tolerate cryopreservation in liquid nitrogen well (Berg et al. 2009, Childs et al. 2013). The problem is less severe in feeder based systems but remains a serious problem for feeder free systems (Berg et al. 2009). Losses in both viability and cytotoxicity resulting from cryopreservation are only partly rescued by addition of IL-2 to culture media (Childs et al. 2013, Berg et al. 2009). Cryopreservation of NK cells is a clinical necessity for adoptive transfer immunotherapy because without it only cells freshly isolated from patient blood can be used. Fresh NK cells require a patient to be ready for infusion at a very specific time point after isolation and if that time point is missed, something that frequently occurs with ill patients, the entire procedure must be aborted.
What is needed is an increased supply of NK cells cultured in feeder free systems available for adoptive transfer procedures. The supply of NK cells would be greatly expanded by the ability to efficiently cryopreserve and then later expand NK cells in vitro and restore cytotoxicity without feeder cells and without requiring large quantities of cytokines. Such NK cells would be available to patients on a more flexible basis and remove a barrier to adoptive transfer of NK cells.